In line with the Ki67 data, the level of TK-1 expression was significantly increased in 14-day AngII AAA tissue than in saline control tissue with a relative difference of approximately 151%, followed by a decrease to a 78%
relative difference in 28-day AngII AAA tissues, supporting the notion that cell proliferation is an event that occurs during the progressive phase of AAA in this model. [18F]FLT is a substrate for cytoplasmic TK-1, which has been demonstrated as a proliferation biomarker in leukaemia, Hodgkin’s and non-Hodgkin’s lymphoma, lung carcinoma, and breast cancer, amongst other cancers (Zhou et al., 2013). Furthermore, studies of TK-1 expression in cancer have revealed its associations with tumour stage, histological grade, metastasis, size, and distant and local recurrence (He et al., 2000, Mao et al., 2002, He et al., 2006, Aufderklamm et al., 2012, Nisman et al., 2013).
The (i) overexpression of TK-1 and (ii) its correlation with the Ki67
proliferation index have also been demonstrated in various cancers (Mao et al., 2005, Chen et al., 2013, Bagegni et al., 2017).
In addition to TK-1, the expression levels of the equilibrative and
concentrative transporters ENT-1, ENT-2, CNT-1, and CNT-3 were found to be significantly increased in 14-day AngII AAA tissue compared to those in saline control tissues with relative differences of 160–185%, followed by a decrease to 45–90% relative differences in 28-day AngII AAA tissues. These transporters contribute to nucleoside homeostasis, and as they are
responsible for the cellular uptake of some nucleoside-based drugs, they may be valuable in nucleoside-derived therapy (Molina-Arcas et al., 2009, Jiraskova et al., 2018). These nucleoside transporters are also reported to influence [18F]FLT uptake; Paproski et al. previously characterised [18F]FLT transport by these transporters in cancer cell lines and demonstrated that they contribute to [18F]FLT uptake, with ENT-1 showing the most significant expression (Paproski et al., 2008, Paproski et al., 2010). There is an
established association between [18F]FLT uptake and proliferation- or thymidine-associated marker expression in various cancers, some of which include Ki67 expression in breast cancer, CD8 and Ki67 expression in metastatic prostate cancer, Ki67 and TK-1 expression in lung cancer, and proliferating cell nuclear antigen and TK-1 expression in fibrosarcoma; these
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associations are interesting to draw parallels with correlations amongst [18F]FLT uptake and the expression of TK-1, ENT-1, ENT-2, CNT-1, and CNT-3 observed in AAA (Barthel et al., 2003, Brockenbrough et al., 2011, Woolf et al., 2014, Scarpelli et al., 2019). Furthermore, concomitant
malignancies have been reported in up to 14% of AAA cases, of which most involve colorectal cancer (Jibawi et al., 2011). Although it might be ambitious to propose that cancers and AAA involve similar proliferative mechanisms, further studies are warranted to determine whether significant commonalities exist between their pathogeneses. Considering our current understanding of [18F]FLT in cancer progression and proliferation, the findings of these
proteins being upregulated and expressed in AngII AAA tissue support the notion of proliferation occurring at an early stage of AAA development.
4.3 [
18F]FLT uptake in the AngII AAA model
Given the significantly high proportion of Ki67-positive nuclei at the end of the period of AngII infusion (28 days post-implantation of the mini-pump), [18F]FLT PET/CT was planned to be performed on day 28. As this
experiment was complex and expensive, the decision was made to perform an additional scan at a second time point (i.e. day 14) to identify if there was a change in radiotracer uptake over time in the AngII AAA disease course. In this study, the uptake of [18F]FLT in AAA was successfully demonstrated for the first time. Interestingly, the uptake significantly decreased by a relative difference of 55% between days 14 and 28 of the model, suggesting that there may be a decrease in proliferative activity in the late stage of AAA.
Furthermore, the alignment of [18F]FLT results with Ki67 staining data and supporting evidence from Western blotting for key players in the [18F]FLT mechanistic pathway support the PET/CT observations and raise the
potential of using [18F]FLT as a tool to observe proliferative activity in in vivo model systems or indeed in humans.
As described in section 1.3.5, AAA formation and progression rely on multiple contributing factors. These include changes in the mechanical properties of the vessel wall, such as wall stress and elasticity; inflammatory cell infiltration of the aortic wall; increased autoimmunity; enhanced oxidative stress; vascular remodelling; degradation of the ECM; and microcalcification.
This multifactorial nature corresponds to many cells contributing to the AAA pathophysiology, such as lymphocytes, mast cells, macrophages, ECM proteins, VSMCs, and endothelial cells, amongst others (Wang et al., 2014, Kuivaniemi et al., 2015, Sun et al., 2018). Reports of the precise cell types that may contribute to the early-stage proliferative signal are largely
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inconclusive, owing to the wide variety of implicated cell types and difficulties in accurately identifying them in vivo using antibody staining. Although the experiments presented in this thesis do not concretely prove that the proliferative signal originates from VSMCs, the findings are in line with reports of VSMC apoptosis, medial wall thinning, and degeneration in late-stage AAA, which are critical for aortic dilatation and rupture
(Lopez-Candales et al., 1997, Henderson et al., 1999, Ailawadi et al., 2009, Riches et al., 2013, Salmon et al., 2013, Clement et al., 2019, Quintana and Taylor, 2019). These events are associated with VSMC phenotypic switching early in AAA development. The reduced density of VSMCs in late-stage AAA is suggested to be the result of apoptosis, evidenced by the observation of apoptotic VSMCs in the medial layer of AAA in humans (Rowe et al., 2000, Kuivaniemi et al., 2015). When the rate of VSMC apoptosis exceeds the rate of VSMC proliferation, the number of SMC layers in the aortic wall
decreases, eventually leading to rupture. When VSMC proliferation is promoted via anti-inflammatory treatment with interleukin-10, the
degradation of SMCs is inhibited, leading to a delay in the development of AAA in rabbits (Zhu et al., 2019). Aneurysm growth is also inhibited in ApoE
-/- mice following treatment with the xanthine derivative KMUP-3, which
inhibits AAA phenotypic switching and apoptosis (Lai et al., 2020). Moreover, studies have demonstrated VSMC proliferation and the role of
dedifferentiated medial VSMCs in neo-intimal development following vascular injury (Herring et al., 2014, Roostalu et al., 2018). These findings propose an important role for VSMCs in AAA development; however, further experiments are warranted to determine the precise contributions of other cell types and regardless of the implicated cell types, it is suggested that the pathobiology differs between early- and late-stage AAA.
The overall findings of an increase in proliferation-associated biomarkers at 14 days followed by a decrease at 28 days point to an active period of cell proliferation early in the AngII-infused AAA disease course that then leads to replicative senescence and reduced proliferative activity late in the disease course. ‘Replicative senescence’ was first described in the context of human fibroblasts in culture and reflects the process that limits the proliferative activity of cells, as cells have a finite life span in which division occurs (Campisi, 1997). Cell senescence has been reported in patients with AAA and patients manifesting risk factors of AAA (Liao et al., 2000, Gacchina et al., 2011), and senescence can also progress to apoptosis (Thompson et al., 1997). In addition, accelerated replicative senescence comprises a distinct phenotype of VSMCs in human AAA compared to that of VSMCs in the
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aneurysmal inferior mesenteric artery, indicating that senescence may play a role in the VSMC reduction that is specifically observed in AAA (Liao et al., 2000, Riches et al., 2018). Furthermore, this late-stage VSMC reduction may reflect the increased accumulation of ECM degradative proteins, the
production of which is stimulated by VSMCs, as well as endothelial cells, adventitial fibroblasts, and inflammatory cells that are adherent to the ECM.
In AAA, the amount of proteins that confer aortic wall integrity, such as
collagen and elastin, is reduced compared to that in normal aortae (Lin et al., 2018, Quintana and Taylor, 2019). An imbalance between proteolytic
enzymes, such as MMPs, and their inhibitors contributes to this reduction;
correspondingly, various MMPs have been shown to be produced by
VSMCs, and a marked increase in MMP expression has then been shown to be associated with a reduction in VSMCs with AAA disease progression (Knox et al., 1997, Mao et al., 1999, Kadoglou and Liapis, 2004, Fanjul-Fernández et al., 2010, Courtois et al., 2013, Lin et al., 2018, Quintana and Taylor, 2019). This imbalance leads to a disruption in the equilibrium
between ECM synthesis and degradation, which then influences the course of expansion and rupture of AAA. For example, slow-growing aneurysms may reflect ECM synthesis mechanisms counterbalancing the ECM
degradation mechanisms. Acceleration of aneurysm growth and rupture in the AAA disease course may result from this equilibrium shifting towards degradation. Therefore, it is encouraging that the data presented in this thesis suggest a period of decreased proliferative activity late in the AAA disease course, which may suggest that VSMCs may partially contribute to the proliferative signal being detected.
Ex vivo gamma counting of whole organs, which provided definitive evidence of the [18F]FLT hotspots noted on the PET/CT images, revealed 160%
greater [18F]FLT uptake in 14-day AngII AAA compared to saline control aortae. All the counted organs also demonstrated greater uptake of [18F]FLT compared to that in the saline control organs (Figure 3.29). This observation may be explained by the effects of AngII on widespread cell proliferation in the AngII-infused model of AAA. As introduced in section 1.4.1.1, AngII mediates growth processes and has been shown to induce the activity of other vasoactive factors, such as endothelin, which further confer growth-altering effects in cells of the kidneys, lungs, and intestines, among other organs (Johnson et al., 1992, Wolf and Wenzel, 2004, Slice et al., 2005, Wang et al., 2015). Regardless of the effects of AngII in other organs, it is encouraging and convincing to observe such a significant increase in
proliferative activity as measured by [18F]FLT uptake in the aneurysmal aorta
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of the classical AngII-infused model of AAA. In addition to the abdominal aorta, the spleen may be another interesting region for analysis. As expected, [18F]FLT uptake was observed in the spleen of all animals.
Significant differences in splenic SUVs were not observed between saline control and 14-day AngII AAA mice. However, in 28-day AngII AAA mice, the splenic SUVs showed a 33% relative difference and a slightly greater SEM.
Further investigation of this splenic uptake in a greater number of 28-day AngII AAA mice is warranted to better understand if long-term AAA disease is associated with changes in proliferative activity in the spleen. The spleen is a major repository of proliferative cells, as monocytes differentiate into dendritic cells and macrophages in tissue healing and repair processes (Drutman et al., 2012); thus, [18F]FLT uptake in the spleen is expected.
Splenic uptake of [18F]FLT was also noted by Ye et al. in atherosclerotic mice, although with a difference in splenic SUVs between wildtype and ApoE-/- mice (Ye et al., 2015). Furthermore, in male patients with AAA, maximal aortic diameter and spleen volume exhibit a strong positive
correlation (Li et al., 2017); however, more data are needed to suggest that spleen enlargement may play a role as an indicator of AAA progression and rupture. The variations in splenic SUVs observed in this study are in line with these previous findings, indicating that further research on the association between splenic activity and AAA progression is warranted.
Finally, a positive correlation was observed between ex vivo [18F]FLT uptake and aortic volume at 14 days. Given the limitations of this study, there was no evidence to suggest that aortic volume remains positively correlated specifically with [18F]FLT uptake in later stages of AAA development, while other mechanisms may contribute to changes in aortic volume with further AAA progression. Nonetheless, this result is important in the context of early-stage AAA management post-USS screening, as [18F]FLT uptake may provide an additional dimension of information about AAA progression:
proliferative activity in relation to early aortic volume changes. The utility of this additional dimension has considerable potential in risk assessment; for example, a small but highly active AAA may warrant treatment, whereas an indolent large AAA may not. Similar to the way in which SUV thresholds of [18F]FDG uptake are currently implemented in the cancer field to classify tumour grade/aggressiveness and guide patient intervention, there may thus be a role for [18F]FLT uptake thresholds in AAA management. Following from the previous example, a patient exhibiting greater aneurysmal [18F]FLT uptake (and thus harbouring early-stage AAA) might benefit more from an anti-proliferative drug therapy, compared to a patient exhibiting lower
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[18F]FLT uptake (and thus harbouring late-stage AAA) who would benefit from surgical repair. Therapy for AAA currently involves patient health optimisation and surgical intervention. An added layer of information may pave the way for trialling medical therapies as opposed to implementing surgery-only options based on physical aneurysm characteristics. However, to confirm the correlation between [18F]FLT uptake in the abdominal aorta and AAA severity, further longitudinal preclinical investigations of
aneurysmal [18F]FLT uptake and its associations with aortic size at baseline, 14 days, and 28 days in the AngII AAA model are needed, a key limiting aspect in the current study due to the high rates of mortality, non-response to AngII, and failure of intravenous radiotracer administration, as well as the significantly high costs of the AngII AAA mouse model. Using the same cohort of mice from study initiation (i.e. day of mini-pump implantation) to completion (i.e. day 28 of AngII infusion) would yield robust results, although large numbers of animals would be needed to mitigate the aforementioned challenges. In terms of clinical research, a large-scale analysis of patient data to precisely determine the relationship between aortic sizes based on USS screening and [18F]FLT uptake on PET would facilitate a clearer understanding of whether [18F]FLT SUVs might be used as informative indicators of size and/or AAA stage.